Jul 31, 2023
Microscopy-based bead protein-protein interaction assay
- 1Max Perutz Labs, University of Vienna, Vienna Biocenter, Vienna, Austria;
- 2Vienna Biocenter PhD Program, Doctoral School of the University of Vienna and Medical University of Vienna, Vienna, Austria
Protocol Citation: Elisabeth Holzer 2023. Microscopy-based bead protein-protein interaction assay. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp3erpvzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 07, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 83013
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: : Mechanisms of mitochondrial damage control by PINK1 and Parkin (ASAP-000350)
Abstract
This protocol describes how to perform microscopy-based bead protein-protein interaction assay with GST- or mCherry-tagged proteins as baits and fluorescently-tagged proteins as preys. The protocol requires to have purified proteins and allows to monitor protein-protein interaction in an equilibrium state. The fluorescent signal can be quantified.
Guidelines
Experiment should be repeated at least three times for statistical analysis.
Materials
Glutathione Sepharose 4B (Cytiva)
RFP-Trap Agarose beads (ChromoTek)
384-well glass-bottom microplate (Greiner Bio-One)
confocal microscope
ImageJ software
Before start
SEC Buffer:
25 mM HEPES pH 7.5
150 mM NaCl
Freshly added: 1 mM DTT
Purify tagged bait and prey proteins
Protocol
NAME
EXPRESSION AND PURIFICATION OF HUMAN NEMO (GST-GFP-NEMO)CREATED BY
OLIVIA HARDING
Prepare bait-coated beads
Prepare bait-coated beads
1h 2m
1h 2m
Equilibrate 20 µL Glutathione Sepharose 4B or RFP-Trap Agarose beads with 200 µL SEC buffer
Note
Prepare these bead equilibrations for each condition.
Incubate equilibrated beads with GST- or mCherry-tagged bait protein for a final concentration of 5 micromolar (µM) in SEC buffer for 01:00:00 at 4 °C with gentle rotation.
1h
Centrifuge beads at 3000 rcf for 00:02:00 at 4 °C
2m
Remove the supernatant and wash beads with 200 µL SEC buffer
Repeat for a total of 2 washes, then discard buffer
Add 20 µL SEC buffer to achieve a beads:buffer ratio of 1:1
Interaction assay set-up
Interaction assay set-up
30m
30m
Pipette prey proteins into the wells of a 384-well glass-bottom microplate (Greiner Bio-One)
Note
A volume of at least 20 µL should be used to cover the bottom of the well
Note
Prey concentration should be 0.1 micromolar (µM) to 1 micromolar (µM) , but should be adjusted depending on the strength of the interaction
Note
Different preys should be conjugated to different fluorophores
Pipette 1 µL of bait-coated beads into each well
Incubate the plate for 00:30:00 in the dark at Room temperature
30m
Signal detection
Signal detection
Use a microscope configured to detect fluorescent signal (e.g. Zeiss LSM 700 confocal microscope equipped with Plan-Apochromat 20X/0.8 objective)
Acquire fluorescent images in the middle section of the beads and collect more than one image for each well
Also acquire bright field images for each field
Quantification using ImageJ
Quantification using ImageJ
Draw several lines across each bead in the fluorescent channel and measure the intensity along the lines
Record the maximum intensity for each bead
For background correction, measure the average intensity of a rectangular ROI that covers an area of each field of view with no beads
Subtract the average intensity of the background ROI from each bead maximum in that field
Calculate the average of the background-corrected maximum intensities of beads for each sample